Dissertation / PhD Thesis

FZJ-2014-02843

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Pupylierung in https $\textit{Corynebacterium glutamicum}$

Küberl, A. (Corresponding Author)FZJ*

2014
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag Jülich
ISBN: 978-3-89336-969-0

Please use a persistent id in citations: http://hdl.handle.net/2128/5847

Abstract: Pupylation represents a post-translational, covalent protein modification by the small protein Pup. Hitherto, this mechanism has been investigated exclusively in actinobacteria, which tag certain proteins with Pup for degradation via the proteasome (Pup proteasome system). The actinobacterium $\textit{Corynebacterium glutamicum}$ possesses the genes coding for the known components of the pupylation machinery ($\textit{pup, pafA, arc}$, and $\textit{dop}$) but not for a proteasome. In the course of the present thesis, it was investigated in $\textit{C. glutamicum}$ whether pupylation is active $\textit{in vivo}$, which proteins are pupylation targets and which functions pupylation exhibits in the absence of a proteasome. Enrichment of poly-histidine-tagged Pup and subsequent MS/MS analyses revealed 55 pupylated proteins in $\textit{C. glutamicum}$, of which 60% belong to the categories „metabolism“ or „translation“. Thus, it could be shown for the first time that $\textit{C. glutamicum}$ possesses a pupylation machinery which is active $\textit{in vivo}$. In addition, an assay for $\textit{in vitro}$ pupylation of proteins was developed using recombinant Pup and the Pup ligase PafA from $\textit{C. glutamicum}$. Hereafter, this assay will provide a tool to confirm pupylation of putative targets and to perform $\textit{in vitro}$ assays with pupylated proteins. The physiological role of pupylation in $\textit{C. glutamicum}$ was investigated by characterization of deletion mutants lacking Pup (Δ$\textit{pup}$), the depupylase Dop (Δ$\textit{dop}$) or the AAA+ ATPase ARC that recognizes pupylated proteins (Δ$\textit{arc}$) under standard growth conditions and iron starvation. All mutants exhibited reduced growth in comparison to the wild type under iron starvation. The Δ$\textit{pup}$ mutant showed differences in the transcriptome and the soluble proteome in comparison the wild type under iron starvation, which can be explained by iron-dependent effects of the transcriptional regulators DtxR and RipA. Investigations of deletion mutants lacking the iron storage proteins ferritin (Δ$\textit{ftn}$) and Dps (Δ$\textit{dps}$) and genes encoding components of the pupylation machinery could show that the growth deficiency of Δ$\textit{pup}$ and Δ$\textit{arc}$ mutants can be complemented by the deletion of the iron storage proteins. Furthermore, the exchange of the pupylated lysine residue in ferritin resulted in a growth defect comparable to the $\textit{pup}$ deletion. Hence, the defects of the mutants can be explained by defective pupylation of iron storage proteins ferritin and Dps. In conclusion, pupylation could represent a novel mechanism to release iron in actinobacteria.

Keyword(s): Dissertation

Note: Heinrich-Heine-Universität Düsseldorf, Diss., 2014

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Contributing Institute(s):

1. Biotechnologie (IBG-1)
2. Biotechnologie 1 (IBT-1)

Research Program(s):

1. 899 - ohne Topic (POF2-899) (POF2-899)

Appears in the scientific report 2014

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Database coverage:

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